Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family.

BackgroundIt is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.ResultsPhylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.ConclusionsLike the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5'-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Members of the Rid protein family have broad imine deaminase activity and can accelerate the Pseudomonas aeruginosa D-arginine dehydrogenase (DauA) reaction in vitro.

The Rid (YjgF/YER057c/UK114) protein family is a group of small, sequence diverse proteins that consists of eight subfamilies. The archetypal RidA subfamily is found in all domains, while the Rid1-7 subfamilies are present only in prokaryotes. Bacterial genomes often encode multiple members of the Rid superfamily. The best characterized member of this protein family, RidA from Salmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate generated by pyridoxal 5'-phosphate-dependent enzymes and ultimately spares certain enzymes from damage. The accumulation of 2-aminoacrylate can damage enzymes and lead to growth defects in bacteria, plants, and yeast. While all subfamily members have been annotated as imine deaminases based on the RidA characterization, experimental evidence to support this annotation exists for a single protein outside the RidA subfamily. Here we report that six proteins, spanning Rid subfamilies 1-3, deaminate a variety of imine/enamine substrates with differing specific activities. Proteins from the Rid2 and Rid3 subfamilies, but not from the RidA and Rid1 subfamilies deaminated iminoarginine, generated in situ by the Pseudomonas aeruginosa D-arginine dehydrogenase DauA. These data biochemically distinguished the subfamilies and showed Rid proteins have activity on a metabolite that is physiologically relevant in Pseudomonas and other bacteria

RidA activity <i>in vivo</i>.

<p>The PLP-dependent generation of the enamine 2 aminoacrylate from serine. Enamine/imine intermediates (2-aminoacrylate/iminopropionate) are in equilibrium and the latter is hydrolyzed by solvent water or facilitated by RidA protein, resulting in production of pyruvate. 2AA accumulation in an <i>S</i>. <i>enterica ridA</i> mutant is responsible for the inactivation of particular PLP-enzymes, which leads to growth defects.</p

Rid proteins deaminate 2AA <i>in vitro</i>.

<p>Reaction mixtures (100 μL) contained Tris-HCl (100 mM, pH 8), NADH (250 μM), pyridoxal 5’-phosphate (30 μM), pyruvate kinase/lactate dehydrogenase (5 Units) and purified cysteine desulfhydrase CdsH (0.27 μM). Purified Rid protein (0.19 μM) was added as indicated. <b>(A)</b> Absorbance was monitored at 340 nm for 60 seconds following the addition of L-cysteine to 1 mM final concentration. Reactions contained CdsH and purified Rid proteins as indicated. <b>(B)</b> The initial rate of pyruvate formation for each reaction was calculated using the molar extinction coefficient (ε = 6,200 M^-1 cm^-1) for NADH oxidation during the first 30 seconds. The mean of three replicates is plotted and error bars indicate standard deviation.</p

Representative RidA and Rid1 proteins rescue growth of <i>S</i>. <i>enterica ridA</i> mutant strain.

<p>An <i>S</i>. <i>enterica ridA</i> strain (DM12920) was transformed with pBAD24 constructs harboring no insert (pBAD24), <i>S</i>. <i>enterica ridA</i>, P. <i>aeruginosa</i> PA0814, <i>A</i>. <i>baylyi</i> ACIAD3089, <i>P</i>. <i>syringae</i> PSPTO_0102, <i>P</i>. <i>aeruginosa</i> PA5083, <i>P</i>. <i>fluorescens</i> PFL_1385, or <i>P</i>. <i>syringae</i> PSPTO_3006. The strains were grown in minimal glycerol medium supplemented with <b>(A-D)</b> 5 mM serine or <b>(E-H)</b> 250 μM cysteine, with (closed symbols) or without (open symbols) arabinose. The corresponding Rid subfamily assignment is presented above each graph. Growth was monitored by optical density at 650 nm with shaking at 37°C. Error bars indicate standard deviation for three biological replicates.</p

Rid2 and Rid3 proteins deaminate iminoarginine produced by DauA.

<p>Assay mixtures (100 μL) were monitored in microtiter plate format and contained potassium pyrophosphate (50 mM, pH 8.7), neutralized semicarbazide (10 mM), bovine liver catalase (24 Units) and ~1 μM DauA (1.7 μM total protein) from the partial purification. <b>(A)</b> Rid protein (10 μM) was added and the substrate was D-arginine (1 mM). Following the addition of substrate, the path length of each well was measured and the change in absorbance at 248 nm was monitored for ten minutes. <b>(B)</b> The bar graph shows the rate of semicarbazone formation calculated from the observed rate of product formation at 248 nm using the molar extinction coefficient (ε = 10,300 M^-1 cm^-1). <b>(C)</b> The rate of semicarbazone formation is presented as a percentage of the rate observed in the control (DauA alone) reaction mixture that lacks Rid proteins. All reaction mixtures contained ~ 1 μM DauA and were initiated with 1 mM D-arginine. The effect of purified Rid2 and Rid3 protein concentration on the rate is shown. Error bars indicate standard deviation of three replicates and curves were fitted using the equation for one phase exponential decay using Prism.</p

Primers used in plasmid construction.

<p>Primers used in plasmid construction.</p

Reaction schemes for FAD-dependent oxidase.

<p>The reaction mechanism shows the FAD-dependent production of an imine from an amino acid. Imines are hydrolyzed by solvent water or facilitated by Rid proteins, or in the presence of semicarbazide react to form a semicarbazone compound. R’ represents the amino acid functional group.</p